Hopkins Et Al. 2020 Cortisol and Giant Salamanders

General and Comparative Endocrinology 285 (2020) 113267

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General and Comparative Endocrinology

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Cortisol is the predominant glucocorticoid in the giant paedomorphic T hellbender salamander (Cryptobranchus alleganiensis) ⁎ William A. Hopkinsa, , Sarah E. DuRantb, Michelle L. Becka,c, W. Keith Rayd, Richard F. Helmd, L. Michael Romeroe a Dept of Fish and Wildlife Conservation, Virginia Tech, Blacksburg, VA 24061, USA b Dept of Biological Sciences, University of Arkansas, Fayetteville, AR 72701, USA c Dept. of Biology, Rivier University, Nashua, NH 03060, USA d Dept of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA e Dept of Biology, Tufts University, Medford, MA 02144, USA

ARTICLE INFO ABSTRACT

Keywords: Corticosterone is widely regarded to be the predominant glucocorticoid produced in amphibians. However, we Amphibian recently described unusually low baseline and stress-induced corticosterone profiles in eastern hellbenders Corticosterone (Cryptobranchus alleganiensis alleganiensis), a giant, fully aquatic salamander. Here, we hypothesized that hell- ACTH benders might also produce cortisol, the predominant glucocorticoid used by fishes and non-rodent mammals. Glucocorticoid To test our hypothesis, we collected plasma samples in two field experiments and analyzed them using multiple Paedomorphosis analytical techniques to determine how plasma concentrations of cortisol and corticosterone co-varied after 1) physical restraint and 2) injection with adrenocorticotropic hormone (ACTH), the pituitary hormone responsible for triggering the release of glucocorticoids from amphibian interrenal glands. Using liquid chromatography- mass spectrometry, we found that baseline and restraint-induced plasma concentrations of cortisol were more than five times those of corticosterone. We then demonstrated that plasma concentrations of both glucocorticoids increased in response to ACTH in a dose-dependent manner, but cortisol concentrations were consistently higher (up to 10-fold) than corticosterone. Cortisol and corticosterone concentrations were not correlated with one another at basal or induced conditions. The extremely low plasma concentrations of corticosterone in hellbenders suggests that corticosterone could simply be a byproduct of cortisol production, and raises questions as to whether corticosterone has any distinct physiological function in hellbenders. Our results indicate that hellbenders produce cortisol as their predominant glucocorticoid, supporting a small and inconclusive body of literature indicating that some other amphibians may produce appreciable quantities of cortisol. We hypothesize that the use of cortisol by hellbenders could be an adaptation to their fully aquatic life history due to cortisol’s ability to fulfill both mineralocorticoid and glucocorticoid functions, similar to its functions in fishes. Giventhe large number of amphibian species that are fully aquatic or have aquatic life stages, we suggest that the broadly held assumption that corticosterone is the predominant glucocorticoid in all amphibians requires further scru- tiny. Ultimately, multi-species tests of this assumption will reveal the ecological factors that influenced the evolution of endocrine adaptations among amphibian lineages, and may provide insight into convergent evolution of endocrine traits in paedomorphic species.

1. Introduction demanding and “stressful” conditions (Greenburg and Wingfield, 1987; Herman, 1992; Norris and Carr, 2013; Wingfield and Romero, 2001; Corticosterone is widely regarded as the predominant glucocorti- Moore and Jessop, 2003). Anurans (frogs and toads) release corticos- coid produced in most amphibians in response to energetically terone when faced with a vast array of environmental stimuli including

Abbreviations: ACTH, adrenocorticotropic hormone; EIA, enzyme immunoassay; HPLC, high-performance liquid chromatography; LC–MS, liquid chromatography mass spectrometry; MDLC, multi-dimensional liquid chromatography; RIA, radioimmunoassay; P4, Progesterone; 17OHP4, 17α-hydroxyprogesterone; DOC, 11- deoxycorticosterone; S, 11-deoxycortisol ⁎ Corresponding author. E-mail address: [email protected] (W.A. Hopkins). https://doi.org/10.1016/j.ygcen.2019.113267 Received 25 June 2019; Received in revised form 19 August 2019; Accepted 1 September 2019 Available online 03 September 2019 0016-6480/ © 2019 Elsevier Inc. All rights reserved. W.A. Hopkins, et al. General and Comparative Endocrinology 285 (2020) 113267 exposure to pollutants (Hopkins et al., 1997), capture and handling 2. Methods (Belden et al., 2010), pathogens (Gabor et al., 2013), pond drying (Denver, 1998), predators (Maher et al., 2013), competition 2.1. Study species (Glennemeier and Denver, 2002a), and food deprivation (Glennemeier and Denver, 2002a; Reeve et al., 2013). In addition, corticosterone Hellbenders are giant salamanders in the Family Cryptobranchidae, modulates energetically expensive anuran calling behaviors during the which only contains two other species globally (Andrias davidianus and reproductive season (Hopkins et al., 1997; Emerson and Hess, 2001; A. japonicus in China and Japan). All three species are considered im- Leary et al., 2006; Moore and Jessop, 2003; Eikenaar et al., 2012) and periled and population trends suggest they could eventually face ex- key developmental events associated with anuran metamorphosis tinction. In North America, there are two recognized subspecies of (Hayes, 1997; Denver, 1998; Wright et al., 2003). Likewise, Urodeles hellbenders; the eastern subspecies (Cryptobranchus alleganiensis alle- (salamanders) release corticosterone in response to an equally diverse ganiensis) and the Ozark subspecies (C. a. bishopi). Both subspecies have array of stimuli (e.g., Gendron et al., 1997; Homan et al., 2003; experienced precipitous population declines in the last few decades Cooperman et al., 2004; Schubert et al., 2009; Hopkins and DuRant, (Wheeler et al., 2003; Briggler et al., 2007; Foster et al., 2009), re- 2011; Ricciardella et al., 2010; Davis and Gabor, 2015; DuRant et al., sulting in the Ozark subspecies receiving federal protection and one 2015; but see Fonner and Woodley, 2015). Although generalizable evolutionarily distinct lineage of the eastern subspecies being con- patterns (e.g., corticosterone as the dominant glucocorticoid in am- sidered for similar status (USFWS 2011a,b, 2019). The last population phibians) are widespread in endocrinology, exceptions to these gen- stronghold for eastern hellbenders is believed to occur across portions eralizations can reveal important areas of ecological and evolutionary of Appalachia. In the area of Virginia where our work was conducted, divergence among taxa. the major threats to hellbender populations are most likely habitat We recently described unusual corticosterone profiles in eastern degradation, disease and parasitism, and direct human persecution hellbenders (Cryptobranchus alleganiensis alleganiensis), the only North (Hopkins and DuRant, 2011; Davis and Hopkins, 2013; DuRant et al., American member of the ancient giant salamander Family 2015; Hopkins et al., 2014; Hopkins et al., 2016; Jachowski and Cryptobranchidae. Hellbenders are obligate paedomorphs that display a Hopkins, 2018). number of unusual physiological, genetic, and morphological traits Hellbenders are large (up to 70 cm total length), long-lived compared to other amphibians (Nickerson and Mays, 1973; Jerrett and (> 30 years) fully aquatic salamanders that thrive in cool, well oxy- Mays, 1973; Sun and Mueller, 2014), but their endocrine physiology genated lotic habitats (Nickerson and Mays, 1973; Taber et al., 1975; remains poorly studied. We demonstrated that this giant salamander Petranka, 1998). Adult and subadult age classes require large cover produces atypically low concentrations of corticosterone in response to objects such as boulders and bedrock crevices, and they feed pre- handling and restraint (Hopkins and DuRant, 2011; DuRant et al., dominantly on crayfish. In most populations, hellbenders are thought to 2015). Baseline concentrations of corticosterone in this species average be primarily nocturnal, but diurnal movement may become more fre- ~0.25 ng/ml and increase to ~1.0 ng/ml after 1–3 h of restraint quent in mid- to late-summer when males become more aggressive and (Hopkins and DuRant, 2011; DuRant et al., 2015). Generally speaking, attempt to commandeer large nest rocks (Noeske and Nickerson 1979; these concentrations are a fraction of those observed in other sala- Humphries and Pauley, 2005) and in the spring following prolonged mander species (Cooperman et al., 2004; Ricciardella et al., 2010; fasting after overwintering (Hopkins, unpublished). Reproduction takes Schubert et al., 2009; Woodley and Lacy, 2010). Understanding whe- place in late August and early September under nest rocks where males ther hellbenders physiologically operate with unusually low con- externally fertilize eggs, and then remain to guard the eggs and larvae centrations of corticosterone, or if they use an alternative glucocorti- until the following April when larvae disperse from the nest (Hopkins, coid, could further our understanding of ecological factors that unpublished). influence the evolutionary divergence of endocrine adaptations among vertebrate lineages. 2.2. Hellbender sampling & treatment Because hellbenders belong to one of the earliest diverging lineages of modern day salamanders (Frost et al., 2006) and exhibit a fully We sampled hellbenders from a stream in the Tennessee River Basin aquatic lifecycle, we hypothesized that their glucocorticoid profiles in Southwest, VA that has been one of our focal study areas since 2007 might more closely resemble an ancestral state that is more similar to (Hopkins and DuRant, 2011; Davis and Hopkins, 2013; DuRant et al., fishes than many other amphibians. Most teleosts produce cortisol 2015; Hopkins et al., 2016; Jachowski and Hopkins, 2018). During which performs both mineralocorticoid and glucocorticoid functions diurnal skin-diving surveys (from 0942 to 1636 h), we collected hell- (Mommsen et al., 1999; Bentley, 2002; McCormick and Bradshaw, benders by turning cover objects and searching crevices with dive 2006; Takahashi and Sakamoto, 2013). This possibility is supported by lights. We conducted all surveys in late summer (31 July – 21 Aug a small body of evidence suggesting that some fully aquatic amphibians 2012; IACUC #11-140 FIW) prior to nesting because at this time adult produce appreciable quantities of cortisol (Phillips and Chester Jones, male and female hellbenders can be distinguished by the swollen cloaca 1957; Chester Jones et al., 1959; Bern and Nandi, 1964; Nandi, 1967; of males (Makowsky, et al., 2010). Once a hellbender was captured, we Norris and Carr, 2013). As an alternative explanation to the ancestral immediately (average time 130 ± 6 s) obtained an initial baseline osteichthyian condition, use of cortisol could be an adaptation to blood sample from the caudal vein using a heparinized 26 gauge needle aquatic life in paedomorphic species, which has independently evolved affixed to a 1 cc syringe (Hopkins and DuRant, 2011). in multiple salamander families including Cryptobranchidae, Proteidae, To verify which steroid hormones increased in response to handling Sirenidae, Ambysotomatidae, Amphiumidae, and Salamandridae (Frost stress, we conducted an initial pilot experiment where 5 adult hell- et al., 2006). Little is known about the glucocorticoid physiology of benders (3 females, 2 males) were restrained in mesh bags submerged species in several of these families. To address our hypothesis that in the stream after the initial blood collection. These individuals were cortisol could be an important glucocorticoid in hellbenders, we con- bled a second time after one hour of restraint. Blood from the initial and ducted two field experiments and then used several analytical techni- post-restraint bleeds was immediately placed on ice and returned to the ques to determine: 1) if cortisol and corticosterone were both produced laboratory where plasma was separated by centrifugation. Plasma from by hellbenders in response to handling, and 2) how these two hormones these individuals was stored frozen (-80° C) and later subjected to liquid varied in concentration after injection with multiple concentrations of chromatography-mass spectrometry (see methods below) to quantify adrenocorticotropic hormone (ACTH), the pituitary hormone re- changes in cortisol and corticosterone due to capture and restraint. This sponsible for triggering the release of glucocorticoids from vertebrate pilot experiment verified that both hormones were present in plasma adrenal glands (a.k.a. interrenal glands in amphibians). (see results), and justified a more in depth exploration of their

2 W.A. Hopkins, et al. General and Comparative Endocrinology 285 (2020) 113267 dynamics. followed by a standard solution (5 μL) containing 75 ng/mL each of d4- For the comprehensive experiment, we transported each hellbender cortisol and d8-corticosterone. We mixed the sample with a precipita- (after the initial blood sample) to the stream bank to be assigned to a tion solution (160 μL) that contained methanol, ethanol, acetone; treatment group and measured. Hellbenders were immediately (1:1:1; v/v/v) and vortexed for 1 min. We centrifuged the resulting weighed, sexed, and then assigned to one of four treatment groups: sample (13,000×g; 10 min) and the supernatant was transferred to a saline [control], 50, 100, or 200 IU kg−1 of ACTH (N = 11 per treat- fresh tube and taken to dryness on a high-vacuum line. We then re- ment group). In total, 44 hellbenders were included in the study. We constituted the sample in water: methanol (50 μL; 80:20, v/v) to be stratified sexes (N = 6–7 females & 4–5 males per treatment) andbody used in LC–MS analysis. sizes (average 40.3–41.5 cm total length in each treatment) across all We injected 10 μL of each sample onto a Jupiter 4 µ Proteo 90 Å four treatment groups. We used intraperitoneal injections of porcine 50 × 0.5 mm reversed phase column (Phenomenex) using a G1313A ACTH (Sigma Chemical A6303) because it provoked adrenal responses 1100 series autosampler (Agilent). The HPLC gradient was developed in previous studies with amphibians and reptiles (e.g. Glennemeier and utilizing a Tempo nano MDLC (Eksigent) and column effluent was in- Denver, 2002b; Romero and Wikelski, 2006), including hellbenders troduced into an 4000 QTrap mass spectrometer (Applied Biosystems) (DuRant et al., 2015). We transported aliquots of different pre-made utilizing a TurboIonSpray electrospray source (Applied Biosystems). stock solutions for each ACTH treatment group to the field on liquid Solvents A and B were LC-MS grade water (Spectrum Chemical) sup- nitrogen. We defrosted aliquots at the time of capture and adjusted plemented with 0.1% (v/v) formic acid and LC–MS grade acetonitrile volumes of injections according to the hellbender’s mass, and used si- (Spectrum Chemical) supplemented with 0.1% (v/v) formic acid, re- milar volumes of saline for the control group (total volume range across spectively. Flow rate was 20 µL/min throughout the entire 30 min treatments = 120–160 μL). To monitor responses to injections, we bled gradient and initial conditions were 80% solvent A and 20% solvent B. hellbenders again at 1- and 3-h post capture by holding them in mesh Initial conditions were maintained for 5 min following injection of the dive bags submerged in shaded areas within the stream near the site of sample. A linear gradient from 80% A to 20% A over 10 min followed. capture. Our prior work demonstrated that peak adrenal responses to The 20% A was maintained for 5 min followed by a linear gradient back restraint and ACTH injection occurred within 3 h of capture (DuRant to 80% A and a re-equilibration at 80% A for 5 min prior to injection of et al., 2015). After completing the third blood draw at 3 hrs post-cap- the next sample. We quantified cortisol and corticosterone utilizing the ture, we released each hellbender under the rock in which it was in- sum of the transition intensities described in Table 1 following nor- itially found. malization to the sum of the transition intensities for the internal Before placing a hellbender into a mesh bag for the three hour re- standards d4-cortisol and d8-corticosterone, respectively, also listed in straint period, we processed each individual according to our pre- Table 1. Data acquisition conditions for the mass spectrometer were: viously described methods. Briefly, we measured (total length and ion spray 5200 V, source temperature 180 °C, interface heater on, CAD snout-vent length) each hellbender and classified them as juvenile or gas medium, entrance potential 10 V. Curtain gas, ion source gas 1, and adult based on size (adult: ≥29 cm total length in our study system; ion source gas 2 were set to 12, 12 and 24, respectively. Hopkins and DuRant, 2011). Only adults were used in this study. We documented the presence/absence of ectoparasites (leeches), because 2.3.2. Corticosterone enzyme immunoassay our previous work demonstrated that parasitism influences hellbender We determined plasma corticosterone levels using enzyme im- physiology (DuRant et al., 2015; Hopkins et al., 2016). We also injected munoassays (EIAs; Cat: No. 900-097, Assay Designs) that were pre- a unique passive integrated transponder tag (PIT tag) into the tail of viously validated and optimized for hellbenders (Hopkins and DuRant, each hellbender as part of our long-term mark recapture efforts. 2011; DuRant et al. 2015). These EIAs have low cross-reactivity with other major steroids such as cortisol (0.046%) according to the manu- 2.3. Hormone analyses facturer. Based on our optimization, we analyzed samples at a 1:3 plasma We used three techniques to measure glucocorticoid hormones. We dilution with 2% steroid displacement buffer. Each 96-well plate in- first used mass spectrometry to measure both corticosterone andcor- cluded a standard curve ranging from 15.63 to 2000 pg/ml with stan- tisol in individuals before and after being subjected to short term re- dards (non-specific binding and total binding) assayed in triplicate. straint. For the larger ACTH experiment, we used an enzyme im- Each plate also contained a 500 pg/ml standard assayed in quad- munoassay (EIA) to measure corticosterone that we had validated in ruplicate to allow us to estimate interplate variability. We ran samples previous studies (Hopkins and DuRant, 2011; DuRant et al., 2015), and in duplicate. Assay detection limit ranged from 0.016 to 0.06 ng/ml. a second EIA that we validated to measure cortisol herein. We also used When plasma corticosterone concentrations fell below the detection mass spectrometry results as an external verification of our EIA results. limit, which occurred in 16 out of 132 samples, we used the plate’s detection limit as the plasma corticosterone concentration for that in- 2.3.1. Liquid chromatography–mass spectrometry (LC–MS) dividual sample. We calculated intra-assay variation as the average We transferred plasma (50 μL) to a 1.5 mL EtOH-washed tube coefficient of variation across all samples within each plate and inter-

Table 1 Mass spectrometer selected reaction monitoring, transition, and instrumental parameters. Retention times: Cortisol = 6.5 min; corticosterone = 11.9 min.

Analyte Q1 Mass (Da) Q3 Mass (Da) Time (msec) DP (V) CE (V) CXP (V)

Corticosterone 347.1 311.0 100 81 23 18 347.1 121.0 100 81 35 20 Cortisol 363.1 121.0 100 81 33 20 363.1 97.0 100 81 51 16 d4-Cortisol 367.1 121.0 100 86 33 20 367.1 97.0 100 86 51 16 d8-Corticosterone 355.1 337.0 100 91 23 20 355.1 125.2 100 93.5 40 14

DP = declustering potential; CE = collision energy; CXP = collision cell exit potential.

3 W.A. Hopkins, et al. General and Comparative Endocrinology 285 (2020) 113267 assay variation across plates as the coefficient of variation of the 500 pg/ml standards. Intra- and inter-assay variation was 3.74% and 3.23%, respectively.

2.3.3. Cortisol enzyme immunoassay We used Enzo Life Sciences EIAs (Cat. 900-071) to quantify cortisol levels in eastern hellbenders. To validate the kits, we quantified cortisol concentrations in the same samples using side-by-side comparisons of mass spectrometry and the kit, and obtained highly concordant cortisol concentrations with the two methods (r = 0.97, p < 0.001; n = 10). In the course of our validation of the EIA, we determined that cortisol concentrations were not quantifiable unless we extracted the samples prior to running the assay. To extract samples, we placed 25 µL of plasma in glass tubes and diluted the sample with 400 µL of distilled water and then vortexed the mixture. We extracted the cortisol by adding 4 mL of dichloromethane to the samples and placed the samples on a shaker for 10 min after which samples sat for 1 h at room temperature without shaking. We removed the lower layer and dried it in a water bath of 38 °C under nitrogen gas until they were dry. Samples were reconstituted in 250 µL of assay buffer and vortexed for the assay. Fig. 1. Plasma concentrations of cortisol and corticosterone (ng/ml) in hell- Average recovery of cortisol from extractions was 61% and we cor- benders at the time of capture and after one hour of restraint. Concentrations rected our final cortisol concentrations based on this extraction effi- were measured from the same 5 individuals using liquid chromatography-mass spectrometry. Values are reported as mean ± 1 SE. ciency. We distributed samples across seven 96 well plates. EIA ingredients were allowed to warm to room temperature for at least 30 min prior to than corticosterone concentrations, respectively. In addition, the cor- running the assay. Each plate contained a standard curve that ranged ticosterone values quantified from mass spectrometry were well aligned from 67.2 to 10,000 pg/ml and each standard curve sample was run in with concentrations of corticosterone that we previously reported using triplicate. We ran a single plasma sample in duplicate on multiple plates EIA and radioimmunoassay methods (Hopkins and DuRant, 2011; in order to assess inter-assay variation that was due to the extraction DuRant et al., 2015). procedure and the variation among plates. All samples were run in In our second field experiment, both plasma corticosterone and duplicate and an additional standard of 3 ng/ml cortisol was run on cortisol concentrations increased due to capture and injection with each plate as well. The assay had a detection limit of 0.085 ng/ml. We saline and ACTH, but cortisol concentrations were an order of magni- calculated intra-assay variation as the average coefficient of variation tude higher than corticosterone at every time point (Fig. 2). In general, between duplicate samples on each plate and inter-assay variation as injection of 50 IU/kg ACTH provoked similar increases in plasma glu- the coefficient of variation among the standards on every plate. Intra- cocorticoids as injection with saline alone (Fig. 2). However, injection assay variation was 13.71% and inter-assay variation was 20.64%. with 100 and 200 IU/kg ACTH induced the release of higher concentrations of both glucocorticoids over time (cortisol: Time: 2.4. Statistical analyses F2,64 = 58.06; p < 0.0001 Treatment × Time: F6,64 = 2.42; p = 0.036; corticosterone: Time F2,61 = 27.54; p < 0.0001 Treat- All statistical analyses were run in Microsoft Excel or SAS 9.1 (SAS ment × Time × Leech F5,61 = 3.29; p = 0.011). The ACTH dose-de- Institute Inc., Cary, NC, USA) and statistical significance was re- pendent temporal dynamics of these changes differed between the two cognized at α < 0.05. Where appropriate, we tested for normal dis- glucocorticoids. Whereas the highest plasma concentrations of corti- tribution of the data and homoscedasticity using Ryan-Joiners and costerone were induced by 200 IU/kg ACTH after one hour, cortisol

Bartlett’s tests, respectively. Corticosterone was log10–transformed to concentrations continued to rise in plasma and were highest at 3 h in better meet assumptions of normality, but raw data were used in other response to the same dose of ACTH. Interestingly, plasma corticos- statistical models. We obtained insufficient plasma from three in- terone and cortisol concentrations did not correlate with each other at dividuals injected with saline to run both hormone analyses; only cor- any time point (in all cases r ≤ 0.10; p ≥ 0.52). ticosterone is reported for these three individuals. To test for the effects of ACTH or saline injection on 4. Discussion log10–corticosterone and cortisol concentrations over time, we per- formed two repeated measures ANOVAs (SAS Proc mixed) that included We demonstrated that plasma concentrations of both corticosterone treatment, leech, and time as main effects and all 2- and 3-way inter- and cortisol increase in hellbenders responding to capture and restraint, actions. We included leech in our models to account for the variation but that cortisol is the predominant glucocorticoid in hellbenders. that they can provoke in hellbender hormone concentrations (DuRant Baseline and restraint-induced plasma cortisol concentrations were > 5 et al., 2015), but a thorough discussion of this effect is reserved for times higher than corticosterone concentrations. Furthermore, injection companion manuscripts (DuRant et al., 2015; DuRant et al., un- with ACTH resulted in dose-dependent increases in both plasma glu- published). We also tested for relationships between corticosterone and cocorticoids, but the induced concentrations of cortisol were again cortisol concentrations at each sampling time point using correlation much higher than corticosterone. In fact, ACTH-injection produced up analysis. to 10-fold higher plasma cortisol concentrations compared to plasma corticosterone concentrations. Such large differences in circulating le- 3. Results vels of the two glucocorticoids suggest that cortisol is likely the predominant glucocorticoid produced by hellbenders, and that corticos- Our liquid chromatography-mass spectrometry analysis confirmed terone’s physiological significance is uncertain. While it is possible that that cortisol is present in much higher concentrations in hellbender the two hormones have different physiological roles (e.g., tissue-spe- plasma than corticosterone (Fig. 1). Specifically, baseline and restraint cific importance in developing songbirds; Schmidt and Soma, 2008), it stress-induced concentrations of cortisol were 5.5 and 5.7 times higher is also possible that corticosterone has negligible importance.

4 W.A. Hopkins, et al. General and Comparative Endocrinology 285 (2020) 113267

Fig. 2. Plasma concentrations of corticosterone and cortisol (ng/ml) in hellbenders at the time of capture and after injections with saline or one of three doses of ACTH (N = 11 per treatment group). Concentrations were measured using EIAs that were verified against results from liquid chromatography-mass spectrometry. Values are reported as mean ± 1 SE. Note difference in scale on y-axes.

Our findings suggest that corticosterone could simply be a metabolic enzyme 17α-hydroxylase. Thereafter, the pathways for cortisol and byproduct of cortisol production in hellbenders (Fig. 3). In other ver- corticosterone production involve identical enzymes; 21-hydroxylase tebrates, the biosynthetic pathways for both glucocorticoids involve converts P4 and 17OHP4 into 11-deoxycorticosterone (DOC) and 11- several identical precursors (i.e., cholesterol, pregnenolone, and pro- deoxycortisol (S), respectively. Similarly, 11β-hydroxylase then con- gesterone) and enzymes (e.g., 21-hydroxylase and 11β-hydroxylase), verts DOC and S into corticosterone and cortisol, respectively. Thus, meaning that production of cortisol inherently requires production of when adrenal stimulation activates the cortisol biosynthesis pathway, corticosterone precursors and activation of corticosterone-producing the subsequent increase in production of shared precursors such as P4 enzymes (reviewed in: Payne and Hales, 2004; Norris and Carr, 2013). and activation of 21-hydroxylase and 11β-hydroxylase could logically Considering that hellbenders exhibit robust production of glucocorti- lead to production of corticosterone as a byproduct (i.e., small amounts coids following treatment with porcine ACTH, it is reasonable to as- of P4 are inadvertently converted to corticosterone while the majority sume that hellbender interrenal physiology follows the general verte- of P4 is processed into 17OP4 for cortisol production). brate paradigm (Fig. 3). Progesterone (P4) is the immediate upstream Interestingly, plasma concentrations of cortisol and corticosterone shared precursor for both cortisol and corticosterone. To produce cor- were not correlated with one another within individuals at any time tisol, P4 is converted into 17α-hydroxyprogesterone (17OHP4) by the point, and show different temporal profiles after hellbenders were

Fig. 3. Biosynthetic pathways of cortisol and corticosterone. Note the shared precursors and enzymes of the two pathways, suggesting the possibility that corticosterone could be the byproduct of cortisol production in hellbenders. Figure Credit: Thomas Galligan, based on Payne and Hales, 2004.

5 W.A. Hopkins, et al. General and Comparative Endocrinology 285 (2020) 113267

Table 2 Summary of studies that have measured both cortisol and corticosterone in amphibians. Detection of each steroid is presented as Detected (Yes) or Not Detected (No).

Species Lifestage Corticosterone Cortisol Predominant GC Method or Source Citation

Xenopus laevis Adult1 No Yes Cortisol Plasma, chromatography Phillips and Chester Jones, 1957 Aphiuma sp. Adult Yes Yes Corticosterone Whole Blood, chromatography Chester Jones et al., 1959 Rana catesbeiana Adult Yes No Corticosterone In vitro interrenal incubation, chromatography Carstensen et al., 1961 Rana sylvatica Larvae No2 Yes Cortisol Urine, chromatography Dale, 1962 Rana pipiens Larvae No2 Yes Cortisol Urine, chromatography Dale, 1962 Bufo marinus Adult No2 Yes Cortisol In vitro interrenal incubation, chromatography Crabbe, 1963 Pleurodeles waltlii Adult Yes No Corticosterone In vitro interrenal incubation, chromatography Ozon and Dupuis-Certain, 1967 Rana catesbeiana Adult Yes No Corticosterone Plasma, chromatography Johnston et al., 1967 Rana rugulosa Adult Yes No Corticosterone In vitro interrenal incubation, chromatography Chan et al., 1969 Xenopus laevis Adult Yes No Corticosterone In vitro interrenal incubation, chromatography Chan and Edwards, 1970 Rana pipiens Adult Yes No Corticosterone In vitro interrenal incubation, chromatography Jungreis et al., 1970 Andrias davidianus Adult Yes No Corticosterone In vitro interrenal incubation, chromatography Chan et al., 1975 Rana catesbeiana Adult Yes No Corticosterone In vitro interrenal incubation, chromatography Mehdi and Carballeira, 1971 Rana catesbeiana Larvae Yes Yes Corticosterone Plasma, radioimmunoassay Krug et al., 1983 Rana catesbeiana Juvenile Yes Yes Cortisol Plasma, radioimmunoassay Krug et al., 1983 Rana catesbeiana Adult Yes Yes Cortisol Plasma, radioimmunoassay Krug et al., 1983 Xenopus laevis Larvae Yes No Corticosterone In vitro interrenal incubation, chromatography Jolivet-Jaudet and Lelop-Hatey, 1984 Xenopus laevis Juvenile Yes No Corticosterone In vitro interrenal incubation, chromatography Jolivet-Jaudet and Lelop-Hatey, 1984 Ambystoma tigrinum Larvae Yes Yes Corticosterone Plasma, radioimmunoassay Stiffler et al., 1986 Ambystoma tigrinum Adult Yes Yes Corticosterone Plasma, radioimmunoassay Stiffler et al., 1986 Rana pipiens Adult Yes No Corticosterone Plasma, radioimmunoassay Stiffler et al., 1986 Triturus carnifex Adult Yes Yes ~Equivalent Plasma, radioimmunoassay Zerani and Gobbetti, 1991 Triturus carnifex Adult Yes Yes ~Equivalent In vitro interrenal incubation, radioimmunoassay Zerani and Gobbetti, 1991 Rana esculenta Adult Yes Yes ~Equivalent Plasma, radioimmunoassay Gobbetti and Zerani, 1993 Rana esculenta Adult Yes Yes ~Equivalent In vitro interrenal incubation, radioimmunoassay Gobbetti and Zerani, 1993 Rana catebeiana Larvae Yes Yes Corticosterone Plasma, radioimmunoassay Wright et al., 2003 Rana catebeiana Adult Yes Yes Corticosterone Plasma, radioimmunoassay Wright et al., 2003 Cryptobranchus alleganiensis Adult Yes Yes Cortisol Plasma, LC–MS and enzyme immunoassay Current Study

1 Study does not provide lifestage, but we assume animals were adult because plasma was analyzed. 2 Study states they measured corticosterone, but no concentration was reported. Thus, we assume it was not detected. injected with ACTH. Whereas corticosterone decreased in plasma 3 h patterns among species with different life histories (Table 2). Not only post-injection with 100 and 200 IU/kg ACTH, cortisol concentrations are results of these studies mixed, but many of them rely on in vitro were still rising three hours after injection with the same two ACTH incubation of interrenal tissue with exogenous precursors to yield glu- doses. These observations suggest that while more cortisol was being cocorticoids. Although these in vitro techniques confirm the glandular secreted into circulation, corticosterone production was either de- presence of enzymes important for steroidogenesis, they do not ne- creasing or it was being cleared from circulation. Similar lack of cov- cessarily reflect the relative hormone concentrations that ultimately ariation between these two glucocorticoids has been described in sev- circulate in plasma. Additional comparative studies that measure eral species of mammals that also primarily rely on cortisol (Koren plasma concentrations of both cortisol and corticosterone in amphi- et al., 2012). There are several possible mechanisms through which this bians are needed, ideally using LC–MS to eliminate cross-reactivity is- could arise. For example, 17α-hydroxylase activity could steadily in- sues associated with antibody-based assays (Koren et al., 2012). crease over the treatment period (3 hrs), leading to a progressive in- It is possible that the use of cortisol by hellbenders is an adaptation crease in the rate at which P4 is metabolized into 17OHP4, effectively associated with paedomorphosis, and that other paedomorphic species reducing the quantity of P4 available for corticosterone production exhibit similar endocrine characteristics. The switch in salamanders to concomitantly with increased cortisol production (Fig. 3). Alter- fully-aquatic life stages or obligate paedomorphism has occurred in- natively, cortisol might exhibit a greater affinity for hellbender corti- dependently multiple times (Bonett and Blair, 2017). Interestingly, re- costeroid binding globulin (CBG) compared to corticosterone, allowing cent research indicates paedomorphic amphibians share several con- cortisol to displace bound corticosterone in circulation, which pro- vergent traits, including body morphology and expression of and gressively accelerates corticosterone clearance. Enzyme activity and insensitivity to thyroid hormones (Bonett and Blair, 2017; Aran et al., CBG affinity studies would be required to test these hypotheses and 2014). The use of cortisol as the primary glucocorticoid could also be fully understand these observed temporal dynamics. the result of convergent evolution in paedomorphic species because it Although the overwhelming majority of previous research shows could provide advantages to an aquatic life style, but support for this that corticosterone is the predominant glucocorticoid in amphibians, hypothesis based on a small number of obligate and facultative pae- our work supports a small and inconclusive body of literature that domorphic species is also mixed (Table 2), including conflicting evi- identifies cortisol production in some amphibian species (Table 2). It is dence between paedomorphic species within Cryptobranchidae (cur- has been postulated that the use of cortisol rather than corticosterone rent study and Chan et al., 1975). Our findings suggest that additional might be associated with the fully aquatic lifestyle of a small number of work is required to verify the importance of cortisol across aquatic species and life stages (Norris and Carr, 2013). For example, there is amphibian species and life stages, especially in paedomorphic species some evidence that fully aquatic paedomorphic species such as Am- and in early divergent amphibian lineages. phiuma produce appreciable quantities of cortisol (Chester Jones et al., The adaptive advantage of cortisol over corticosterone in hellben- 1959; Bern and Nandi, 1964; Nandi, 1967). Likewise, aquatic adult ders remains unknown, but it may be attributable to its ability to reg- stages of the Italian crested newt (Triturus carnifex) produce nearly ulate plasma ion concentrations in aquatic species. Cortisol binds to equivalent concentrations of both cortisol and corticosterone (Zerani both the mineralocorticoid and glucocorticoid receptors in other ver- and Gobbetti, 1991). However, a review of available evidence from tebrates (mammals: Pippal and Fuller, 2008; fishes: Takahashi and studies that have measured both cortisol and corticosterone in amphi- Sakamoto, 2013) and is known to have important ionoregulatory roles bians yields equally contradictory evidence, and thus inconclusive in some vertebrates (Mommsen et al., 1999; Bentley, 2002; McCormick

6 W.A. Hopkins, et al. General and Comparative Endocrinology 285 (2020) 113267 and Bradshaw, 2006; Takahashi and Sakamoto, 2013). For example, References cortisol appears to be the predominant hormone regulating both mineralocorticoid and glucocorticoid functions in fishes, and they tend to Aran, R.P., Steffen, M.A., Martin, S.D., Lopez, O.I., Bonett, R.M., 2014. Reduced effectsof lack appreciable quantities of aldosterone, the principle miner- thyroid hormone on gene expression and metamorphosis in a paedomorphic Plethodontid Salamander. J. Exp. Zool. Part B 332B, 294–303. alocorticoid in terrestrial tetrapods (Mommsen et al., 1999; Bentley, Belden, L.K., Wingfield, J.C., Kiesecker, J.M., 2010. Variation in the hormonal stress re- 2002; McCormick and Bradshaw, 2006; Takahashi and Sakamoto, sponse among larvae of three amphibian species. J. Exp. Zool. Part A 313A, 524–531. 2013). Thus, it seems possible that cortisol plays dual mineralocorticoid Bentley, P.J., 2002. Endocrines and osmoregulation: a comparative account in vertebrates. Vol 1 (2nd ed.). In: Zoophysiology Vol 39. Bradshaw, S.D., Burggren, W., and glucocorticoid functions in hellbenders and some other fully Heller, H.C., Ishii, S., Langer, H., Neuweiler, G., Randall, D.J. (Eds.), Springer-Verlag, aquatic amphibians (Norris and Carr, 2013). Future comparative work Berlin. of cortisol, corticosterone, and aldosterone dynamics among terrestrial Bern, H.A., Nandi, J., 1964. Endocrinology of poikilothermic vertebrates. Hormones 4, and aquatic lineages of amphibians could help elucidate whether re- 199–293. Bonett, R.M., Blair, A.L., 2017. Evidence for complex life cycle constraints on salamander liance on cortisol is in fact an ionoregulatory adaptation to aquatic life. body form diversification. Proc. Nat. Acad. Sci. 114, 9936–9941. Briggler, J.T., Utrup, J., Davidson, C., Humphries, J., Groves, J., Johnson, T., Ettling, J., Wanner, M., Traylor-Holzer, K., Reed, D., Lindgren, V., Byers, O., 2007. Hellbender 4.1. Conclusions population and habitat viability assessment. IUCN/SSC Conservation Breeding Specialist Group, Apple Valley, MN. Given the plight of hellbender populations in recent decades, un- Carstensen, H., Burgers, A.C., Li, C.H., 1961. Demonstration of aldosterone and corticosterone as the principal steroids formed in incubates of adrenals of the American derstanding their glucocorticoid physiology will be important to their bullfrog (Rana catesbeiana) and stimulation of their production by mammalian conservation. Glucocorticoid responses to environmental conditions are adrenocorticotropin. Gen. Comp. Endocrinol. 1, 37–50. a key tool used by conservation physiologists in efforts to monitor in- Chan, S.T.H., Edwards, B.R., 1970. Kinetic studies on the biosynthesis of corticosteroids in vitro from exogenous precursors by the interrenal glands of normal, corticotrophin- dividual health, especially with species of conservation concern like the treated and adenohypophysectomized Xenopus laevis tadpoles. J. Endocrinol. 47, hellbender (Wasser et al., 1997, 2000; Wikelski and Cooke 2006; Cook, 183–195. 2012; Cooke et al., 2013; Narayan, 2013; McCormick and Romero, Chan, S.T.H., Vinson, G.P., Phillip, J.G., 1969. Pathways for the biosynthesis of corticosteroids from pregnenlone by adrenal tissue of the frog, Rana rugulosa. Gen. Comp. 2017). Our use of LC–MS provides the most conclusive evidence to date Endocrinol. 12, 644–650. of an amphibian species relying on cortisol as their predominant glu- Chan, S.T.H., Sandor, T., Lofts, B., 1975. A histological, histochemical, and biochemical cocorticoid. Our work demonstrated that cortisol should be measured in study of the adrenal tissue of the Chinese giant salamander (Andrias davidianus Blanchard). Gen. Comp. Endocrinol. 25, 509–516. future efforts to understand how hellbenders, and perhaps some other Chester Jones, I., Phillips, J.G., Holmes, W.N., 1959. Comparative physiology of the early diverging and/or paedomorphic amphibians, respond to en- adrenal cortex. Comp. Endocrinol. 1959, 582–612. vironmental changes such as compromised water quality. Our LC–MS Cook, N.J., 2012. Review: minimally invasive sampling media and the measurement of work confirmed our cortisol results from the EIA reported herein, as corticosteroids as biomarkers of stress in animals. Can. J. Anim. Sci. 92, 227–259. Cooke, S.J., Sack, L., Franklin, C.E., Farrell, A.P., Beardall, J., Wikelski, M., Chown, S.L., well as our corticosterone results from our previous studies that used 2013. What is conservation physiology? Perspectives on an increasingly integrated EIA (Hopkins and DuRant, 2011; DuRant et al., 2015), and thus verified and essential science. Conserv. Physiol. 1, 1–23. the utility of these two EIAs for future studies of hellbenders. Given the Cooperman, M.D., Reed, J.M., Romero, L.M., 2004. The effects of terrestrial and breeding densities on corticosterone and testosterone levels in spotted salamanders, extremely low plasma concentrations of corticosterone in hellbenders, Ambystoma maculatum. Can. J. Zool. 82, 1795–1803. however, it is possible that corticosterone is simply a byproduct of Crabbe, J., 1963. Nature and metabolism of adrenocortical secretion in the toad (Bufo cortisol production. Future work should determine whether corticos- marinus). Gen. Comp. Endocrinol. 3, 692–693. Dale, E., 1962. Steroid excretion by larval frogs. Gen. Comp. Endocrinol. 2, 171–176. terone has any physiological importance in hellbenders, and whether Davis, A.K., Hopkins, W.A., 2013. Widespread trypanosome infections in a population of cortisol influences multiple physiological processes such as iono-, eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) in Virginia, USA. osmo-, and blood glucose regulation. Moreover, additional work is Parasitol. Res. 112, 453–456. Davis, D.R., Gabor, C.R., 2015. Behavioral and physiological antipredator responses of the needed to determine whether the patterns we observed in hellbenders San Marcos salamander, Eurycea nana. Physiol. Behav. 139, 145–149. occur in other early amphibian lineages and/or species that have Denver, R.J., 1998. Hormonal correlates of environmentally induced metamorphosis in evolved a paedomorphic lifecyle. the Western Spadefoot Toad, Scaphiopus hammondii. Gen. Comp. Endocr. 110, 326–336. DuRant, S.E., Hopkins, W.A., Davis, A.K., Romero, L.M., 2015. Evidence of ectoparasite- Declaration of Competing Interest induced endocrine disruption in an imperiled giant salamander, the eastern hellbender (Cryptobranchus alleganiensis). J. Exp. Biol. 218, 2297–2304. Eikenaar, C., Husak, J., Escallón, C., Moore, I.T., 2012. Variation in testosterone and The authors declare that they have no known competing financial corticosterone in amphibians and reptiles: relationships with latitude, elevation, and interests or personal relationships that could have appeared to influ- breeding season length. Am. Nat. 180, 642–654. ence the work reported in this paper. Emerson, S.B., Hess, D.L., 2001. Glucocorticoids, androgens, testis mass, and the en- ergetics of vocalization in breeding male frogs. Horm. Behav. 39, 59–69. Fonner, C.W., Woodley, S.K., 2015. Testing the predation stress hypothesis: behavioural Acknowledgements and hormonal responses to predator cues in Allegheny Mountain dusky salamanders. Behaviour 152, 797–819. Foster, R.L., McMillan, A.M., Roblee, K., 2009. Population status of hellbender sala- We are grateful for the field assistance of B. Coe, D. Drewitt, C. manders (Cryptobranchus alleganiensis) in the Allegheny River Drainage of New York Jachowski, A.W. Carter, M. Hepner, the laboratory assistance of J. State. J. Herpetol. 43, 579–588. Frost, D.R., Grant, T., Faivovich, J.N., Bain, R.H., Haas, A., Haddad, C.F.B., De Sá, R.O., Jervis and I.T. Moore, editorial assistance of J. Wagner, and helpful Channing, A., Wilkinson, M., Donnellan, S.C., Raxworthy, C.J., Campbell, J.A., discussion of our results and review comments of T. Galligan and I.T. Blotto, B.L., Moler, P., Drewes, R.C., Nussbaum, R.A., Lynch, J.D., Green, D.M., Moore. Two external reviewers provided excellent suggestions the im- Wheeler, W.C., 2006. The amphibian tree of life. Bulletin American Museum of Natural History. Number 297, 370 pp. New York, NY. proved the paper. This work was made possible with the financial Gabor, C.R., Fisher, M.C., Bosch, J., 2013. A non-Invasive stress assay shows that tadpole support of the Fralin Life Sciences Institute at Virginia Tech and the populations infected with Batrachochytrium dendrobatidis have elevated corticos- National Science Foundation (IOS-1755055). All work was done in terone levels. PLoS ONE 8, e56054. accordance with appropriate state scientific collecting permits and an- Gendron, A.D., Bishop, C.A., Fortin, R., Hontela, A., 1997. In vivo testing of the functional integrity of the corticosterone-producing axis in mudpuppy (Amphibia) exposed to imal care protocols. chlorinated hydrocarbons in the wild. Environ. Toxicol. Chem. 16, 1694–1706. Glennemeier, K.A., Denver, R.J., 2002a. Role for corticoids in mediating the response of Rana pipiens tadpoles to intraspecific competition. J. Exper. Zool. 292, 32–40. Appendix A. Supplementary data Glennemeier, K.A., Denver, R.J., 2002b. Developmental changes in interrenal respon- siveness in anuran amphibians. Integr. Comp. Biol. 42, 565–573. Supplementary data to this article can be found online at https:// Gobbetti, A., Zerani, M., 1993. Prostaglandin E2 and Prostaglandin F2α Involvement in the corticosterone and cortisol release by the female frog, Rana esculenta, during doi.org/10.1016/j.ygcen.2019.113267.

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